Insulin and IL-4 sensitivity was restored to the IL-3-dependent 32D cell line by expression of insulin receptor substrate-1 (IRS-I). the principal phosphotyrosine substrate of the insulin receptor. IL-4 treatment of 32D cells overexpressing IRS-I causes prompt tyrosine phosphorylation of IRS- I. Transfection of truncation mutants of the human IL-4 receptor into 32D.IRS-1 cells demonstrated that the region between amino acids 437 and 557 is critical for IL-4 signaling. This region of the IL-4 receptor contains the I4R motif, found in the insulin and IGF-1 receptors. Mutation of Y497 to F yielded receptors that failed to cause IRS-1 phosphorylation in response to human IL-4 when expressed in 32D.IRS-1 cells. Most cell lines expressing Y497F also failed to proliferate in response to human IL-4. furthermore. a GST-fusion protein containing the I4R motif bound IRS-I. a tyrosine kinase(s) and other unidentified phosphoproteins with molecular sizes of 140. 80 and 55 kDa. These results indicate that the central tyrosine of the I4R motif is critical for IL-4-mediated signal transduction in 32D cells. The SH2 domain of GRB-2 specifically binds to a tryptic peptide containing tyrosine 895 of tyrosine phosphorylated IRS-I. Substitution of tyrosine 895 with phenylalanine (IRS-1F895) prevents formation of the IRS-1/GRB-2 complex in vitro and in vivo. Insulin-stimulated mitogenesis in 32D cells requires expression of the insulin receptor and either IRS-I or IRS-1F895. suggesting that the IRS-1/GRB-2 complex is not essential for this response. In contrast. insulin stimulation of Shc tyrosine phosphorylation. formation of the Shc/GRB-2 complex and activation of mitogen activated protein (MAP) kinase required expression of the insulin receptor. but was independent of IRS-I and unaffected by expression of IRS-1F895. Therefore. IRS-I. but not an IRS-1/GRB-2 complex. is required for insulin-stimulated mitogenesis in 32D cells. whereas Shc/GRB-2 may be sufficient for insulin stimulation of MAP kinase through the SOS/p2I ras pathway. However. insulin is not able to mediate an efficient mitogenic response in 32D cells overexpressing only the insulin receptor. suggesting that activation of the SOS/p2I ras pathway may be necessary but not sufficient for eliciting proliferation in this stem.